CN103881936A - Preparation method of solid-state microecological preparation for bacillus natto and bacillus subtilis culture - Google Patents
Preparation method of solid-state microecological preparation for bacillus natto and bacillus subtilis culture Download PDFInfo
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- CN103881936A CN103881936A CN201210555979.XA CN201210555979A CN103881936A CN 103881936 A CN103881936 A CN 103881936A CN 201210555979 A CN201210555979 A CN 201210555979A CN 103881936 A CN103881936 A CN 103881936A
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Abstract
The present invention discloses a preparation method of a solid-state microecological preparation for bacillus natto and bacillus subtilis culture. The preparation method comprises three steps such as liquid bacterial liquid culture, solid base material preparation and uniform solid base material and liquid bacterial liquid mixing, and specifically comprises: mixing a solid base material and a liquid bacterial liquid according to a weight percentage ratio of 1:1, uniformly stirring, loading in a dish, placing into a sterile culture chamber, maintaining a constant temperature of 40 DEG C, carrying out ventilation, and carrying out shallow dish fermentation for 36 h, wherein the surface and the inner layer of the shallow dish are full of the growing white bacterial; and increasing the temperature to 60 DEG C, increasing wind flow, drying the discharged material, determining the live bacteria concentration of 1*10<9> cfu/g, and carrying out crushing sub-packaging to obtain the product. The preparation method has characteristics of simple and easy-control preparation process, impurity bacterial contamination resistance, high strain concentration, low post-processing cost, and environmentally friendly production environment.
Description
Technical field
The invention belongs to solid-state micro-ecological preparation method, be specifically related to the solid-state micro-ecological preparation method of a kind of bacillus natto and subtilis.
Background technology
That micro-ecological product has is nontoxic, have no side effect, and the feature such as noresidue, pollution-free, does not develop immunity to drugs, and cost is lower, is desirable Substitutes For Antibiotic.At present commercially available probiotics has liquid and solid-state two kinds.The solid-state probiotics particularly formulation method of the micro-ecology of composite solid of bacillus natto and subtilis has two kinds of process technology schemes conventionally, and one is first to carry out liquid submerged fermentation, finally adds solid-state carrier absorption, dry forming; Though and liquid state fermentation is cultivated and can be obtained purer thalline, cost is high, fermentation is subject to product feedback control, is difficult to obtain high density bacterial strain, so that the effective bacterium of the method is counted content is lower; Another kind is the pattern of conventional solid state fermentation; But existing these technical matters method ubiquities processing step complexity, and preparation cost is high, the shortcomings such as product yield is low, miscellaneous bacteria content height.
Summary of the invention
The problems referred to above that exist in order to overcome prior art field, the object of the invention is to, provide a kind of bacillus natto and subtilis solid-state micro-ecological preparation method, solve preparation method's complexity of the composite solid probiotics of current bacillus natto and subtilis, the problem that product yield is low, miscellaneous bacteria content is high.
The solid-state micro-ecological preparation method of bacillus natto provided by the invention and subtilis, it comprises three large steps, mixing of the cultivation of liquid bacteria liquid, the preparation of solid base, solid base and liquid bacteria liquid, concrete steps are: the ratio that is 1:1 by weight percentage solid base and liquid bacteria liquid is mixed, sabot after stirring, enter Sterile culture room, 40 ℃ of constant temperature, ventilate, shallow tray fermentation 36 hours, visible tray surface and internal layer all cover with white thalline, improve temperature to 60 ℃, add Wind Volume, dry discharging, records viable bacteria concentration 1 × 10
9cfu/g, pulverizes packing, obtains product.
The cultivation of described liquid bacteria liquid comprises following concrete steps:
One, dull and stereotyped cultivation: bacillus natto and subtilis preserve bacterial classification streak inoculation to plate culture medium, its substratum batching is glucose 6g, peptone 2g, extractum carnis 3g, sodium-chlor 5g, manganous sulfate 5mg, agar 15g, distilled water 1000ml, pH value is 7,121 ℃ of sterilizings, cultivate 30h for 32 ℃;
Two, test tube inoculation: get and grow healthy and strong, the typical inoculation 10ml of bacterium colony, its substratum batching is glucose 6g, peptone 2g, extractum carnis 3g, sodium-chlor 5g, manganous sulfate 5mg, distilled water 1000ml, pH value is 7,121 ℃ of sterilizings, cultivates 24h for 35 ℃;
Three, inoculate 100ml with triangular flask respectively, the same step 2 of its substratum, 121 ℃ of sterilizings, cultivate 12h for 35 ℃;
Four, inoculate 1000ml with triangular flask respectively, the same step 3 of its substratum;
Five, cultivate with 10L seed fermentation tank, nutrient solution is 7L, the same step 3 of substratum, and pH value is 7,121 ℃ of sterilizings 30 minutes, culture temperature is 37 ℃, rotating speed 150r/min, air flow 3vvm, refers to the fermentation volume m of the per minute unit of passing into
3volume of air m
3, cultivate 18h;
Six, cultivate with 100L seed fermentation tank, nutrient solution is 100L, and substratum is pressed bean cake powder 2g, fish meal 2g, 120 object Semen Maydis powder 0.5g, glucose 3g, sodium-chlor 0.2g, manganous sulfate 0.3g, synergistic agent 0.1g, the ratio preparation of water 1000ml, cultivates by the method for step 5, and selected synergistic agent is that zinc sulfate, ferric sulfate and copper sulfate form by equivalent proportioning;
Seven, 1000L fermentor tank, substratum is respectively with the proportioning septuple of 100L seed fermentation tank in step 6, and the same step 5 of fermentation process, cultivates 24h.
Described solid base is by becoming below assignment system: Pericarppium arachidis hypogaeae or cotton seed hulls 500kg, Semen Maydis powder 150kg, bean cake powder 200kg, wheat bran 100kg, glucose 5kg, gross weight is 1000kg, adds boiling sterilization after 200kg water-wet after stirring and evenly mixing, to be cooled during to 35 ℃, add the fermented liquid 700kg in the culturing step seven of liquid bacteria liquid.
The solid-state micro-ecological preparation method of bacillus natto provided by the invention and subtilis, its beneficial effect is, and preparation technology of the present invention is simple and be easy to control, and is difficult for dying miscellaneous bacteria, and bacterial strain concentration is high, and post-treatment cost is low, production environment close friend; Beans genus bacillus is aerobic bacteria, and subtilis is also aerobic bacteria, and both all can grow under aerobic condition, so micro-ecological product can not only effectively supplement the beneficial microorganism in livestock and poultry alimentary tract, and improves archenteric flora balance.The secondary metabolite of these two kinds of bacterium, as somatomedin, the energy enhancing body disease resistance such as antibacterial peptide, improve absorbing of metabolism and feed, thereby reach control digestive tract diseases and promote the multiple actions such as growth.
Embodiment
Below in conjunction with embodiment, to bacillus natto provided by the invention and the solid-state micro-ecological preparation method of subtilis, be described in detail.
Embodiment
The solid-state micro-ecological preparation method of the bacillus natto of the present embodiment and subtilis, it comprises three large steps, mixing of the cultivation of liquid bacteria liquid, the preparation of solid base, solid base and liquid bacteria liquid, concrete steps are: the ratio that is 1:1 by weight percentage solid base and liquid bacteria liquid is mixed, sabot after stirring, enter Sterile culture room, 40 ℃ of constant temperature, ventilate, shallow tray fermentation 36 hours, visible tray surface and internal layer all cover with white thalline, improve temperature to 60 ℃, add Wind Volume, dry discharging, records viable bacteria concentration 1 × 10
9cfu/g, pulverizes packing, obtains product.
The cultivation of described liquid bacteria liquid comprises following concrete steps:
One, dull and stereotyped cultivation: bacillus natto and subtilis preserve bacterial classification streak inoculation to plate culture medium, its substratum batching is glucose 6g, peptone 2g, extractum carnis 3g, sodium-chlor 5g, manganous sulfate 5mg, agar 15g, distilled water 1000ml, pH value is 7,121 ℃ of sterilizings, cultivate 30h for 32 ℃;
Two, test tube inoculation: get and grow healthy and strong, the typical inoculation 10ml of bacterium colony, its substratum batching is glucose 6g, peptone 2g, extractum carnis 3g, sodium-chlor 5g, manganous sulfate 5mg, distilled water 1000ml, pH value is 7,121 ℃ of sterilizings, cultivates 24h for 35 ℃;
Three, inoculate 100ml with triangular flask respectively, the same step 2 of its substratum, 121 ℃ of sterilizings, cultivate 12h for 35 ℃;
Four, inoculate 1000ml with triangular flask respectively, the same step 3 of its substratum;
Five, cultivate with 10L seed fermentation tank, nutrient solution is 7L, the same step 3 of substratum, and pH value is 7,121 ℃ of sterilizings 30 minutes, culture temperature is 37 ℃, rotating speed 150r/min, air flow 3vvm, refers to the fermentation volume m of the per minute unit of passing into
3volume of air m
3, cultivate 18h;
Six, cultivate with 100L seed fermentation tank, nutrient solution is 100L, and substratum is pressed bean cake powder 2g, fish meal 2g, 120 object Semen Maydis powder 0.5g, glucose 3g, sodium-chlor 0.2g, manganous sulfate 0.3g, synergistic agent 0.1g, the ratio preparation of water 1000ml, cultivates by the method for step 5, and selected synergistic agent is that zinc sulfate, ferric sulfate and copper sulfate form by equivalent proportioning;
Seven, 1000L fermentor tank, substratum is respectively with the proportioning septuple of 100L seed fermentation tank in step 6, and the same step 5 of fermentation process, cultivates 24h.
Described solid base is by becoming below assignment system: Pericarppium arachidis hypogaeae or cotton seed hulls 500kg, Semen Maydis powder 150kg, bean cake powder 200kg, wheat bran 100kg, glucose 5kg, gross weight is 1000kg, adds boiling sterilization after 200kg water-wet after stirring and evenly mixing, to be cooled during to 35 ℃, add the fermented liquid 700kg in the culturing step seven of liquid bacteria liquid.
Claims (3)
1. a bacillus natto and the solid-state micro-ecological preparation method of subtilis, it is characterized in that: it comprises three large steps, mixing of the cultivation of liquid bacteria liquid, the preparation of solid base, solid base and liquid bacteria liquid, concrete steps are: the ratio that is 1:1 by weight percentage solid base and liquid bacteria liquid is mixed, sabot after stirring, enter Sterile culture room, 40 ℃ of constant temperature, ventilate, shallow tray fermentation 36 hours, visible tray surface and internal layer all cover with white thalline, improve temperature to 60 ℃, add Wind Volume, dry discharging, records viable bacteria concentration 1 × 10
9cfu/g, pulverizes packing, obtains product.
2. the solid-state micro-ecological preparation method of bacillus natto according to claim 1 and subtilis, is characterized in that: the cultivation of described liquid bacteria liquid comprises following concrete steps:
One, dull and stereotyped cultivation: bacillus natto and subtilis preserve bacterial classification streak inoculation to plate culture medium, its substratum batching is glucose 6g, peptone 2g, extractum carnis 3g, sodium-chlor 5g, manganous sulfate 5mg, agar 15g, distilled water 1000ml, pH value is 7,121 ℃ of sterilizings, cultivate 30h for 32 ℃;
Two, test tube inoculation: get and grow healthy and strong, the typical inoculation 10ml of bacterium colony, its substratum batching is glucose 6g, peptone 2g, extractum carnis 3g, sodium-chlor 5g, manganous sulfate 5mg, distilled water 1000ml, pH value is 7,121 ℃ of sterilizings, cultivates 24h for 35 ℃;
Three, inoculate 100ml with triangular flask respectively, the same step 2 of its substratum, 121 ℃ of sterilizings, cultivate 12h for 35 ℃;
Four, inoculate 1000ml with triangular flask respectively, the same step 3 of its substratum;
Five, cultivate with 10L seed fermentation tank, nutrient solution is 7L, the same step 3 of substratum, and pH value is 7,121 ℃ of sterilizings 30 minutes, culture temperature is 37 ℃, rotating speed 150r/min, air flow 3vvm, refers to the fermentation volume m of the per minute unit of passing into
3volume of air m
3, cultivate 18h;
Six, cultivate with 100L seed fermentation tank, nutrient solution is 100L, and substratum is pressed bean cake powder 2g, fish meal 2g, 120 object Semen Maydis powder 0.5g, glucose 3g, sodium-chlor 0.2g, manganous sulfate 0.3g, synergistic agent 0.1g, the ratio preparation of water 1000ml, cultivates by the method for step 5, and selected synergistic agent is that zinc sulfate, ferric sulfate and copper sulfate form by equivalent proportioning;
Seven, 1000L fermentor tank, substratum is respectively with the proportioning septuple of 100L seed fermentation tank in step 6, and the same step 5 of fermentation process, cultivates 24h.
3. the solid-state micro-ecological preparation method of bacillus natto according to claim 1 and subtilis, it is characterized in that: described solid base is by becoming below assignment system: Pericarppium arachidis hypogaeae or cotton seed hulls 500kg, Semen Maydis powder 150kg, bean cake powder 200kg, wheat bran 100kg, glucose 5kg, gross weight is 1000kg, after stirring and evenly mixing, add boiling sterilization after 200kg water-wet, to be cooled during to 35 ℃, add the fermented liquid 700kg in the culturing step seven of liquid bacteria liquid.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104256183A (en) * | 2014-10-10 | 2015-01-07 | 青岛嘉瑞生物技术有限公司 | Dairy cow heat stress-resistant Chinese herbal fermented feed additive |
| CN106701652A (en) * | 2017-01-04 | 2017-05-24 | 嘉兴国龙生物科技有限公司 | Process for producing biological packing bacterium bacillus natto |
| CN107254423A (en) * | 2017-07-03 | 2017-10-17 | 成都市农林科学院 | A kind of method of preparation and use of Nattokinase microbial inoculum |
| CN111733117A (en) * | 2020-08-17 | 2020-10-02 | 中国科学院烟台海岸带研究所 | Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof |
| CN112293627A (en) * | 2019-11-25 | 2021-02-02 | 重庆康康康生物科技有限公司 | Vegetable protein functional peptide beverage and preparation method thereof |
-
2012
- 2012-12-20 CN CN201210555979.XA patent/CN103881936A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104256183A (en) * | 2014-10-10 | 2015-01-07 | 青岛嘉瑞生物技术有限公司 | Dairy cow heat stress-resistant Chinese herbal fermented feed additive |
| CN106701652A (en) * | 2017-01-04 | 2017-05-24 | 嘉兴国龙生物科技有限公司 | Process for producing biological packing bacterium bacillus natto |
| CN106701652B (en) * | 2017-01-04 | 2020-10-27 | 嘉兴国龙生物科技有限公司 | Production process of biological padding bacterium bacillus natto |
| CN107254423A (en) * | 2017-07-03 | 2017-10-17 | 成都市农林科学院 | A kind of method of preparation and use of Nattokinase microbial inoculum |
| CN107254423B (en) * | 2017-07-03 | 2020-04-07 | 成都市农林科学院 | Preparation and application method of nattokinase microbial inoculum |
| CN112293627A (en) * | 2019-11-25 | 2021-02-02 | 重庆康康康生物科技有限公司 | Vegetable protein functional peptide beverage and preparation method thereof |
| CN111733117A (en) * | 2020-08-17 | 2020-10-02 | 中国科学院烟台海岸带研究所 | Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof |
| CN111733117B (en) * | 2020-08-17 | 2020-12-08 | 中国科学院烟台海岸带研究所 | Bacillus marinus for producing antibacterial peptide and fermentation method and application thereof |
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Application publication date: 20140625 |